Vol. 48 No. 4/2001

829–850

QUARTERLY

Review

Structure of small G and their regulators

0½ Marcin Paduch, Filip Jeleñ and Jacek Otlewski

Institute of Biochemistry and Molecular Biology, University of Wroc³aw, Wroc³aw, Poland Received: 6 November, 2001; accepted: 27 November, 2001

Key words: small G , GTPase, Ras, Rho, GAP, GEF, GDI, protein tertiary structure, protein–protein interaction

In recent years small G proteins have become an intensively studied group of regula- tory GTP involved in signaling. More than 100 small G proteins have been identified in eucaryotes from protozoan to human. The small superfamily includes Ras, Rho , Rac, Sar1/Arf and homologs, which take part in numerous and diverse cellular processes, such as expression, cytoskeleton re- organization, organization, and vesicular and . These proteins share a common structural core, described as the G domain, and significant sequence similarity. In this paper we review the available data on G domain structure, together with a detailed analysis of the mechanism of action. We also present small G protein regulators: GTPase activating proteins that bind to a catalytic G domain and increase its low intrinsic activity, GTPase dissociation inhibitors that stabi- lize the GDP-bound, inactive state of G proteins, and exchange fac- tors that accelerate nucleotide exchange in response to cellular signals. Additionally, in this paper we describe some aspects of small G protein interactions with down- stream effectors.

0 J.O. was supported by an International Scholar’s award from the Howard Hughes Medical Institute. ½ Corresponding author: Jacek Otlewski, Institute of Biochemistry and Molecular Biology, University of Wroc³aw, Tamka 2, 50-137 Wroc³aw, Poland; tel. (48 71) 375 2824; fax. (48 71) 375 2608; e-mail: [email protected] Abbreviations: Arf, ADP ribosylation factor; BH, breakpoint-cluster-region domain; CRD, cystein rich domain; DH, Dbl homology domain; FTIR, Fourier-transform infrared spectroscopy; GAP, GTPase-activating protein; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide ex- change factor; GppCp, guanosine-5¢-(b,g-methylene)-triphosphate; GppNp, guanosine-5¢-(b,g-imido)- g ¢ g b triphosphate; GTP S, guanosine-5 -( -thio)-triphosphate; ICE, interleukin 1 -convertase; Kd, dissocia- tion constant; koff, dissociation rate constant; kon, association rate constant; PH, pleckstrin homology domain; LFER, linear free energy relationships; Pi, inorganic ; PI3-, phosphatidyl inositol 3-kinase; PtdIns(3,4)P2, phosphatidylinositol-3,4-bisphosphate; RBD, Ras binding domain; RGS, regulator of G protein signaling; 830 M. Paduch and others 2001

Small G proteins (also known as small GENERAL PROPERTIES OF SMALL , small GTP binding proteins and Ras G PROTEINS ) form an independent superfamily within the larger class of regula- Numerous comparisons of amino-acid se- tory GTP hydrolases. This superfamily is quences of small G proteins from various spe- made up of a diverse range of molecules that cies have revealed that they are conserved in control a vast number of important processes primary structures at the level of 30–55% and possess a common, structurally preserved homology. Individual members of the Ras GTP-binding domain [1]. Small G proteins are family share relatively high, about 50%, monomeric molecules with masses in the amino-acid sequence identity, whereas Rab range from 20 to 40 kDa, that bind and hydro- and Rho families show about 30% sequence lyze guanine [2]. Although G pro- identity with Ras family members. All G pro- teins are poor catalysts, they can form stable teins possess a consensus sequence responsi- complexes both with the (GTP) and ble for GTP/GDP binding and GTPase hydro- the product (GDP). The GTP and GDP-bound lytic activity [4]. forms define the active and inactive state of The biological function of small G proteins the molecule, respectively. The binding and strongly depends on postranslational modifi- hydrolysis of guanine nucleotides cause sig- cations. For example, Ras, Rho and Rab fami- nificant conformational changes within so lies contain a C-terminal sequence which un- called the switch regions I and II of the pro- dergoes posttranslational modifications tein G catalytic domain. which are essential for the cellular localiza- A wide range of available three-dimensional tion of the proteins and their interaction with structures reveals the basis of protein G regu- effectors. There are several possibilities of lation and activity. The transition between the C-terminal region modifications, as described active and inactive states is aided by regula- in Fig. 1. Probably the most important cova- tory proteins of at least three distinct fami- lent modification is the attachment of lies. The low intrinsic GTPase activity is accel- isoprene moieties and lipids, together with erated by the binding of the GTPase activating methylation and proteolytic cleavage [5–7]. protein (GAP) which influences the duration Posttranslational modifications have not yet of the GTP-bound form. The release and sub- been well characterized, and it is of high inter- sequent rebinding of guanine nucleotide is est to study possible in- promoted by guanine nucleotide exchange fac- hibitors that could be used as drugs in G pro- tor (GEF). Finally, the ability to bind the tein-associated disease treatment [5, 8–10]. effector is abolished in the presence of gua- nine dissociation inhibitor (GDI). There are about 150 known eukaryotic G DOMAIN small G proteins which have been character- ized and divided into five families: Ras, Rho, Crystallographic and NMR analysis of small Rab, Sar1/Arf and Ran [3]. Members of these G proteins, including Ha-Ras, Rho, Rac1, families perform numerous cellular func- Rap2A, Rab3A, Rab7, Arf, Ran, Cdc42 re- tions: Ras and Rho families are involved in vealed the presence of a 20 kDa catalytic do- gene expression, Rho proteins control cyto- main that is unique for the whole superfamily. skeleton reorganization, Rab and Sar1/Arf The domain is built of five a helices (marked families influence vesicular transport, and A1–A5), six b-strands (B1–B6) and five Ran G proteins regulate nuclear transport polypeptide loops (G1–G5) (Fig. 2A). Con- and the cell cycle. trary to a general rule that within different Vol. 48 Small G proteins and their regulators 831

Figure 1. Posttranslational modifications of small G proteins. C-terminal regions can be divided into five groups: A. H-Ras and N-Ras are palmitoylated and farnesylated, a short C-terminal peptide is proteolytically removed (-AAX) and the C-terminal Cys residue is carboxymethylated. B. K-Ras is modified in the same manner as H-Ras and N-Ras but does not possess the additional palmitoylated Cys res- idue, a polybasic region preceding the C-terminal Cys is present. C. is geranylgeranylated, a short peptide (-AALeu/Phe) is removed, the polybasic region and carboxymethylation are also present. D. In Rab3a both Cys resi- dues are geranylgeranylated and the C-terminal Cys is carboxymethylated. E. Cys-Cys seqence of Rab1 is geranylgeranylated and the C-terminal Cys is carboxymethylated. (OCH3), indicates methylation; +, polybasic re- gion; *, linker; A, aliphatic amino acid; X, any amino acid; P, palmitoyl; F, farnesyl; G, geranylgeranyl. protein folds secondary structure elements and Walker B boxes that are found in other are more conserved than loop regions, the nucleotide binding motifs, not homologous to most conserved structural elements of the G small G proteins, present in sugar , domain are the loops (Table 1) [2, 11]. A struc- ABC transporters and ATP [15]. tural comparison of the GTP- and GDP-bound The G2 loop connects the A1 helix and the B2 form, allows one to distinguish two functional strand and contains a conserved Thr residue loop regions: switch I and switch II that sur- responsible for Mg2+ binding. The guanine round the g-phosphate group of the nucleotide base is recognized by the G4 and G5 loops. (Fig. 2) [12, 13]. Switch III region, which is The consensus sequence NKXD of G4 loop present in the a subunits of heterotrimeric G contains Lys and Asp residues directly inter- proteins, is absent in small G proteins [14]. acting with the nucleotide. Part of the G5 loop The G1 loop (also called the P-loop) that con- located between B6 and A5 acts as a recogni- nects the B1 strand and the A1 helix is respon- tion site for the guanine base (Fig. 3) [16]. A sible for the binding of a- and b-phosphate comparison of two complexes, Ras×GDP× groups. The G3 loop provides residues for Mg2+ and Ras×Mg2+, containing a non- Mg2+ and g-phosphate binding and is located hydrolyzable GTP analogue, GppNp or at the N-terminus of the A2 helix. The G1 and GppCp, reveals the presence of two regions G3 loops are sequentially similar to Walker A that undergo structural changes upon GTP 832 M. Paduch and others 2001

Table 1. of conserved loops.

The most conservative amino-acid residues within the G1–G5 loops are indicated in colour, also consensus se- quences are given. (SWISSPROT accession codes for all sequences are listed in the second column.).

G1 (P-loop) G2 (switch I) G3 (switch II) G4 G5 Protein Seqence GXXXXGK(S/T) XTX DXXG (N/T)(K/Q)D (T/G/C)(C/S)A H-Ras P01112 GAGGVGKS YDPT IED I LDTAGQE VGNKCD YIETSAK RhoA P06749 GDGACGKT YVP TVFE L WDTAGQE VGNKKD YMECSAK Cdc42 P25763 GDGAVGKT YVP TVFD L F DTAGQE VGTQ I D YVECSAL Rac1 P15154 GDGAVGKS YIPTVFD L WDTAGQE VGTKLD YLECSAL

Gia P04898 GAGE SGKS RVKTTGI L F DVGGQR FL NKKD THFTCAT EF-Tu P20001 GHVDHGKT RGI TINT HVDGP GHA FL NKCD IVRGSAL Arf-1A P32889 GLGAAGKS TIPT IGF VWDVGGQD FANKQD IQATCAT

binding and hydrolysis (Fig. 2). One of them is movement between Tyr32 in the G2 loop and switch I, which corresponds to the G2 loop phosphate groups [18]. The dynamics of and is called the effector loop because it is a switch I and II differs for GTP and GDP bound site for effector and GAP binding [17]. Switch forms of Ras proteins [19, 20]. Rho, with its II is formed by the G3 loop and part of the A2 minimal catalytic apparatus, maintains the helix and structurally is the most flexible ele- ability to hydrolyze GTP and convert the re- ment of the catalytic domain. leased free energy into a conformational

Figure 2. A schematic diagram of the G domain. 2+ A. Ras×GppCp×Mg complex (PDB 5p21). Five a-helices are marked A1–A5, six b-strands B1–B6 and five polypeptide loops G1–G5. B. Ras×GDP×Mg2+ complex (PDB 4q21). Conformational changes upon GTP hydrolysis can be observed in the switch I and switch II regions.

The dynamics of both switch regions has change in effector regions. These changes are been examined using NMR, EPR and FTIR sufficient for the cycling between the effector- spectroscopy. The proposed “two state” move- bound and effector-free form. The flexibility ment of the effector loop was based on a clear of the G1, G2, and G4 loops is defined as re- Vol. 48 Small G proteins and their regulators 833 gional polysterism that is crucial for the func- ence of Mg2+ on GDP binding is different for tioning of small G proteins [21]. various small G proteins. Mg2+ binds to Ras The differences in G domain structure with micromolar affinity and decreases the among small G proteins mainly concern dissociation rate constant (koff) for GDP by changes in the nucleotide binding region and four orders of magnitude [25]. The interaction structural elements including an additional between the g-phosphate group and Mg2+ in- N-terminal a-helix (present in Rho proteins) duces rigidity of switch I/II, in contrast, GTP or an antiparallel b-sheet in the switch I and II hydrolysis destabilizes the effector region. Al- region (found in Arf and Ran proteins) and though the segments responsible for nucleo- differences in coordination of tide binding are almost identical among G pro- (present in Arf proteins). In Rac a 13 amino- teins, the halftime of GTP/GDP exchange is acid insertion is present and forms an addi- different and varies from minutes to days. At tional a-helix. As an example of the differ- present it is hard to rationalize these substan- ences in the switch II region, RhoA possesses tial kinetic differences. an additional a-helix [21]. An additional Coordination of Mg2+ is mediated by a single b-hairpin in Arf-1A is responsible for function- water molecule and the conserved Asp residue ally relevant oligomerization, which is a rare from the G3 loop (Fig. 3). Alpha-helices sur- phenomenon among G proteins. Displace- rounding the nucleotide are simi- ment of the consensus DXXG sequence in G2 larly essential as well as four amides from the loop by two additional amino acids decreases backbone chain of the G1 loop that form a hy- the ability of Arf proteins to hydrolyze GTP drogen bonding network with oxygens of the [22]. The A1 helix in Ran proteins forms two a and b . The weaker binding ob- separate A1a and A1b helices [23]. Although served for GMP results from single (or at these differences are significant, they do not most double) hydrogen bond formation. The change the overall G protein topology, as de- proper conformation of the G1 loop is accom- scribed [24]. plished by the presence of several glycines In conclusion, a structural comparison of G that adopt torsion angles forbidden for other proteins shows that the G1 phosphate recogni- amino acids (Fig. 3). The G1 loop is well or- tion loop and the G4 nucleotide recognition dered in all crystal structures and is not af- loop are conserved, unlike the G2 Mg2+ coor- fected by GTP hydrolysis. In the majority of dination loop and the G3 g-phosphate binding cases a single substitution of Gly for other res- loop, which are more diverse. Conformational idues in the G1 loop significantly lowers the differences in the loops directly affect the abil- ability of Ras to hydrolyze GTP. Mutations of ity of different G proteins to bind a broad Lys and Ser/Thr residues that are also pres- spectrum of effectors and the kinetics of GTP ent in the G1 loop affect mainly GTP/GDP hydrolysis. binding, since the Lys side chain links a and b phosphate groups, and the hydroxyl group of Ser (or Thr) governs coordination of Mg2+ GUANINE NUCLEOTIDE RECOGNITION (Fig. 3) [26, 27]. — SPECIFICITY OF INTERACTION A G protein in its free form (without the nu- cleotide) has a significantly decreased stabil- In all three-dimensional structures G pro- ity and determination of its affinity for nucle- teins are associated with Mg2+, which is essen- otide binding is inherently difficult. There- tial for proper GTP/GDP binding as it coordi- fore, nucleotide affinity is usually estimated nates oxygen atoms of the b- and g-phosphate according to koff values of the G protein groups. In vitro studies 100 nM Mg2+ is suffi- complexed with a nucleotide. In Ras proteins –5 cient to ensure nucleotide binding. The influ- the affinity is higher for GTP (koff about 10 834 M. Paduch and others 2001

Figure 3. The guanine nucleotide binding site. Ras×GppCp×Mg2+ complex (PDB 5p21). Side chains of important residues are coloured and labeled with the one-letter amino-acid code.

–1 –4 –1 s ) than for GDP (koff about 10 s ) [28]. only in the G1, G2 and G3 loops but also in a These data together with a rough estimate of segments well outside the loop regions. the association rate constant (kon) for the free Intramolecular interactions within the B1 and form of Ras provide an estimation of Kd value B2 loops and the A5 helix influence the stabil- at the level of 10–11 M–1. The affinity for ity of the G5 loop and are responsible for the guanosine alone or for GMP is about six or- affinity toward GDP [19, 20]. Upon GTP hy- ders of magnitude lower than for GDP/GTP drolysis the structural elements that are re- [29]. Binding of the nucleotide to Ras is asso- sponsible for binding of g-phosphate and Mg2+ ciated with a local folding event of slow kinet- undergo dramatic changes. Coordination con- ics [19, 20]. The Mg2+ and g-phosphate bind- tacts of Mg2+ with the anion of the g-phos- ing is governed by the two most flexible ele- phate and the hydroxyl of Ser (Thr) in the ments of the G domain: the G2 (switch I) and effector loop are lost. Also Gly of the G3 loop G3 (switch II) loops. In the known complexes, looses its contact with the g-phosphate of the Mg2+ is hexacoordinated to the b- and g-phos- GTP. These lost contacts are replaced by an phate groups, to Ser (or Thr) from the G1 interaction of Mg2+ with water molecules. In loop, to Thr from the G2 loop, and to two wa- some G proteins the interaction between an ter molecules [27]. Additionally, the g-phos- Asp residue from the G3 loop and Mg2+ is me- phate of GTP forms a hydrogen bond with the diated by a water molecule. Disruption of this Gly residue in the DXXG sequence of the G3 interaction during GTP hydrolysis decreases loop, and with the hydroxyl and amide of the the affinity for Mg2+ in the GDP bound state. Ser (Thr) residue in the G1 loop (Fig. 3). In A lower affinity for Mg2+ is a permanent fea- Ras proteins an additional hydrogen bond is ture of Arf-1A, where five of seven coordina- formed by Tyr32 hydroxyl and g-phosphate tion spheres are filled with water molecules [18]. [22]. Site-directed mutagenesis studies of the G The Asn and Asp residues from the NKXD domain showed that many amino-acid resi- sequence of the G4 loop participate in the hy- dues contribute to the affinity toward guanine drogen bonding with the guanine ring. The nucleotides. These residues are located not aliphatic part of the Lys side chain contacts Vol. 48 Small G proteins and their regulators 835 the purine ring, whereas its e-amino group bonyl in the G2 loop (Fig. 4A) [35]. The crystal forms a regular hydrogen bond with the structures of the oncogenic Ras protein, endocyclic oxygen atom of the ribose (Fig. 3). which has a Gln to Glu mutation and is inac- The exocyclic keto oxygen in position 6 of the tive, together with a positive effect on kcat of guanine ring is hydrogen bonded to one of the the reverse Glu to Gln mutation seem to con- main-chain amides in the G5 loop. Loss of this firm the model. Accordingly, Gln seems to be interaction reduces the affinity by two to too weak base to accept a proton. However, three orders of magnitude, thus providing the Gln residue is capable of stabilizing the strong discrimination against adenine nucleo- pentavalent intermediate state in the hydroly- tides [30]. It is accomplished by the lack of fa- sis reaction (Fig. 4B) [36]. The pH dependence vorable bonding of the Asp and Asn residues of the activity reveals the presence of in NKXD to the adenine ring. Mutation of ei- a functional group of pKa about 3 that is im- ther of these residues drastically decreases portant in hydrolysis. The sharp transition in the affinity for guanosine or switches the chemical shifts for the enzyme with bound 31P specificity with serious physiological conse- points to the g-phosphate group of GTP [37]. quences [31–33]. GTP binds better to G pro- Indeed, exchange of Mn2+ for Mg2+ acceler- teins than ATP by seven orders of magnitude. ates the hydrolysis rate via increasing the abil- The purine ring stabilization mechanism var- ity of g-phosphate to accept the proton [38]. ies among small G proteins. In the case of Ras, This type of reaction is described as sub- a Phe residue in close proximity to the G2 loop strate-assisted catalysis [39]. While the pro- or amino acids from the G5 loop serve this ton transfer step may not correspond to the ki- function as well as hydrogen bonding between netic activation barrier, it can be the rate lim- hydroxyl groups of ribose ring and main-chain iting factor [40]. carbonyl or side-chain carboxylate groups Based on a detailed theoretical analysis of close to the G2 region [34]. crystal and NMR structures as well as LFER analysis of Ras mutants two models describ- ing the intermediate state of GTP hydrolysis MECHANISM OF GTP HYDROLYSIS have been proposed (Fig. 4E). The dissociative model assumes that the first catalytic event is Small G proteins are very inefficient hydro- the disruption of the bond between b- and –1 g lases with kcat in the range from 0.03 min -phosphate. In effect, an unstable meta- (for Ras) to 0.003 min–1 (for EF-Tu and phosphate intermediate is formed with a Arf-1A), i.e. about 1% of an average reaction charge transition from b-tog-phosphate. The rate of heterotrimeric G proteins [2]. GTP hy- associative model assumes the presence of the drolysis proceeds according to the SN2 mecha- pentavalent intermediate and accumulation nism that stands for direct transfer of the of a negative charge on the g-phosphate. This g GTP -phosphate group to H2O with inversion event is directly followed by the hydrolysis of of configuration around the phosphate atom. the bond between b and g-phosphate with a The central role in catalysis is supposed to be charge transition to b-phosphate (Fig. 4C) × 2+× – – played by a Gln residue from the G3 loop [41]. GDP Mg AlF4 (or AlF3OH ) com- (Fig. 4). This residue acts as a catalytic base to plexes are stable transition state analogs that activate a water molecule for the nucleophilic maintain the ability to interact with GAP. The – attack. In crystal structures the water geometry of the AlF4 complex is identical in molecule is located at the distance no longer the pentacoordinate intermediate state [35, than 4 Å from the g-phosphate group and is 42]. Based on crystal structures of the ana- oriented for the nucleophilic attack due to hy- logs, the carboxyamide group of Gln (G3 loop) drogen bonding with g-phosphate and car- is able to polarize and orient the nucleophilic 836 M. Paduch and others 2001

Figure 4. Mechanism of GTP hydrolysis, according to Berghuis et al. [35]. A.Gia1×GTPgS×Mg2+ enzyme–substrate complex (PDB 1gia). A water molecule lies 3.8 Å from the g-phosphate g × × –× 2+ and is positioned for a nucleophilic attack. R178 and Q204 do not contact GTP S [47]. B. Gia1 GDP AlF4 Mg – b pentacoordinate intermediate (PDB 1gfi). The AlF4 forms octahedral complex where the -phosphate and a water molecule are placed in transaxial positions. R178 can form hydrogen bonds with the pentacoordinate intermediate and the oxygen bridging the phosphates. Such orientation of R178 supports both the associative and the dissociative models of GTP hydrolysis. Q204 and T181 are properly placed so that the attacking water molecule is polarized and × × well oriented [47]. C. Gia1 GDP Pi enzyme–products complex (PDB 1git). Q204 (switch I) moves away and the 2+ Mg coordination sphere is disrupted. R178 forms hydrogen bonds with b-phosphate and Pi. T181 as well as the pre- ceding K180 (not marked) reorient and form hydrogen bonds with Pi, this causes a in switch × I. The most affected region is switch II as a result of the escape of Q204 from the catalytic site [35]. D. Gia1 GDP complex (PDB 1gg2). Switch II is disordered. Switch I moves away from the nucleotide because of the loss of the Mg2+ . The catalytic site is disrupted [48]. E. Two models representing the transition state of the GTP hydroly- sis reaction. water molecule in the transition state for hy- ent which stabilizes the developing negative drolysis (Fig. 4B). In heterotrimeric G pro- charge on the released phosphate group teins an additional Arg in the G2 loop is pres- (Fig. 4C). Proper orientation of the Gln and Vol. 48 Small G proteins and their regulators 837

Arg residues is the reaction rate-limiting fac- needed for effector recognition. GTP hydroly- × 2+× – tor. Upon GDP Mg AlF4 binding these sis causes a change in the favorable conforma- two residues are being correctly oriented and tion and a mobility of the effector interacting help to stabilize the intermediate state both in region. The interaction energy of small G pro- the associative and the dissociative model tein–effector/regulator promotes the transi- (Fig. 4B) [40]. The lack of the catalytic Arg res- tion to the intermediate state and speeds up idue in the small G is mainly re- the hydrolysis [50]. sponsible for their low GTPase activity. The Binding of GTP×Mg2+ maintains the active crucial Arg residue can be delivered by the conformation of the switch I and II regions GAP making hydrolysis much more efficient [19, 20]. The Gly residue in the DXXG se- [43]. There is no known complex of the small quence is essential for reorientation and local – – G protein with AlF4 . The energy of AlF4 folding of this region. A Gly to Ala mutation binding is not sufficient for the formation of blocks the GTP-induced conformational the metastable bipiramidal transition state change and thus seriously decreases the analog due to a deficiency of the stabilizing effector affinity. Reorientation of the Tyr and helical domain or an Arg residue [44]. Thr (ligands for Mg2+) that serve as the An analysis of FITR spectra along with com- g-phosphate binding residues occurs as a re- puter aided calculations enabled a more de- sult of a conformational change in switch I. tailed description of GTP hydrolysis. Upon The changes within both switch regions are GTP binding, reorientation of the Lys16, highly correlated [51]. The dynamic proper- Gly15 and Val14 amides (P-loop) forces the ties of the switch I region are important for flexible GTP molecule into a strained confor- effector binding. Removal of the Thr residue mation [45]. The negative charge of g-phos- coordinating Mg2+ affects the dynamics as phate is then transferred to the b-phosphate well as effector interaction and appears to be group. The charge redistribution results in an responsible for the conservation of this resi- electron structure resemblance of GTP to due in GTP-binding proteins [17]. The region GDP and is the main factor decreasing the en- responsible in G proteins for effector binding ergy of transition activation barrier for the in- has been studied by site-directed mutagene- termediate state of reaction [46]. sis, for example a study of RIP/RalBP1 re- vealed the presence of residues responsible for the discrimination between Ras and Ral INTERACTION WITH EFFECTORS effectors [52]. One of the proteins regulated by Ras is the Ser/Thr kinase Raf-1 which in A large number of proteins is known to be turn interacts with MEKK in the MAP kinases regulated by small G proteins. The most im- pathway. Raf-1 contains an 80 amino-acid Ras portant ones are depicted in Table 2. Small G binding domain (RBD) that binds to Ras and proteins utilize GTP binding energy for stabi- Rap-1A with Kd = 20 nM in a GTP dependent lization of the two switch regions, which is manner [53]. Rap-1A is a homolog of Ras that

Table 2. Selected effectors of the Rho/Cdc42/Rac family, according to Bourne et al. [1] and Aspenstrom [49].

PI3-kinase, PI4,5-kinase, , rhophilin, kinectin, rhotekin, DGKz, PKN, MBS, Rho ROK/ROCK/Rho kinase, Bni1, Bnr1, Pkc1, p140mDia, Fks1, Fks2 NADPH , PI3-kinase, PI4,5-kinase, DGK, PAK, POSH, IQGAP, MLK2, MLK3, Cdc42 POR1, Sra-1, S6-kinase ACK, PAK, PI3-kinase, WASP, N-WASP, S6-kinase, MLK2, MLK3, IQGAP, MRCK, MSE5, Rac Skm1, Gic1, Gic2, Borg, Bni1, Ste20, Cla4 838 M. Paduch and others 2001 activates MAP kinases. The effector loop and gamma catalytic domain and a change in con- the B2 and B3 strands form the kinase Raf-1 formation of the effector may represent an recognition site [54]. RBD possesses a typical allosteric component of the activation ubiquitin fold. The b-strands B2 in RBD and (Fig. 5B) [56]. B2 in Rap-1 form an antiparallel b-sheet that Modifiers of the interaction of G proteins directly serves as an interaction site (Fig. 5A). with effectors are also known. A protein Formation of an intermolecular b-sheet is a named 14-3-3 seems to interact with the Raf-1 common mechanism for all known RBDs [55, kinase and supports Ras in its activation [59]. 56]. Additionally, the C-terminal Arg residue Another protein that helps Ras in transducing of the A1 helix in RBD is crucial for small G the signal to its effectors is KSR (kinase sup- protein binding. Interestingly, switch II and pressor of Ras) [60]. This modifier facilitates other GTP recognition residues in Rap-1A do signal transmission between Raf, MAP and not take part in the interaction with RBDRaf-1 MAP kinase [61]. The CNK protein (connector [57]. In RBDRap-1A complex switch II is sol- enhancer of KSR) possesses several pro- vent exposed and is able to bind a modulator tein–protein interaction domains (SAM, PDZ,

Figure 5. Interaction of small G proteins with effectors. The RBD domains form intermolecular b-sheets with the switch I region at the interaction site. A. Raf1RBD×Gp- pNHp×Rap1A complex (PDB 1gua). B. H-Ras×PI3K gamma complex. (PDB 1he8). protein such as RasGAP. Binding of RasGAP PH and proline rich region) and acts up- to Raf-1×Ras×GTP×Mg2+ induces GTP hydro- stream of Raf modifying signal propagation lysis followed by a release of Raf-1 from the [62]. complex. The Zn2+ binding cysteine-rich do- main (CRD) forms a second site of Ras bind- ing in Raf-1 [58]. RBD domains can also be MOLECULAR SWITCHES AND THE found in the group of small G protein modula- ACTIVATION-INACTIVATION CYCLE tors such as RalGEF [55]. A loop in the RBD domain of phospho- inositide 3-kinase gamma (PI3K gamma) posi- The signaling pathways mediated by small G tions Ras so that it uses both the switch I and proteins are widely recognized as responsible switch II regions to interact with the effector. for regulation of many processes in eukary- Ras also forms a direct contact with the PI3K otic cells. Many membrane receptors and up- Vol. 48 Small G proteins and their regulators 839 stream regulators are known that activate these pathways [63]. The outstanding diver- sity of cellular targets that can interact with small G proteins has recently been reviewed [64]. It is also known that there is coope- rativity and coactivation among various mem- bers of the small G protein superfamily and their regulators. For example, the cross-talk between Ras and Rho proteins involves two classes of regulatory mechanisms. One of them is signal divergence, in which a signal from one activates two different types of GEFs: Ras GEF and Rho GEF. Alter- natively, there is a common GEF protein, which is able to activate both the Ras and Rho Figure 6. Mode of activation of small G protein proteins. Signal convergence is an outcome of superfamily. activation of one effector molecule by two dif- GAPs stimulate the low GTPase activity to promote the ferent small G proteins resulting in a single conversion of the GTP-bound to the GDP-bound form. cellular response [65]. GDIs affect dissociation of GDP and maintain the GTPases in their inactive state. GEFs after receiving Low GTPase activity and GDP dissociation the signal catalyze the exchange of GTP for GDP. rate are of ultimate physiological importance since they enable precise positive and nega- tive regulation of small G proteins (Fig. 6). brane and the cytosol seems to be physiologi- This regulation is mainly accomplished by cally more important than the inhibition of GTPase dissociation inhibitors (GDI), GTPase nucleotide dissociation itself. activating proteins (GAP) and guanine nucleo- Up to date, two types of GDI proteins have tide exchange factor proteins (GEF). In the ac- been described: RabGDI and RhoGDI (Fig. 7). tive, GTP-bound state, GTPases interact with Interestingly, even though there is no struc- target proteins to promote a cellular re- tural similarity between RhoGDI and sponse. GEF catalyzes the exchange of GTP RabGDI, they perform the same function in for bound GDP. The low intrinsic GTPase ac- different families of small G proteins. RabGDI tivity is stimulated by GAP and completes the regulates membrane association and recy- cycle. Additional regulation is provided by cling of the Rab family of small G proteins, GDI that keeps the G protein in the GDP- which are critical in vesicle-membrane traf- bound, inactive, state (Fig. 6). ficking. The 50 kDa a isoform of RabGDI is built of two domains: the larger b domain, composed of seven antiparallel and seven par- GDI allel b-strands, and the smaller a helical do- main. The smaller domain is structurally simi- GDI proteins bind to a variety of proteins lar to monooxygenases and flavine . that are postranslationally modified by addi- The b domain of RabGDI contains the Rab tion of the geranylgeranyl moiety. GDIs are binding GCD domain and a conserved region able to extract these proteins from the cellular that are both responsible for the interaction membrane and create their cytosolic pool. with geranylgeranyltransferase II. The GCD Cytosolic G proteins occur mainly in the domain is similar to the Rep protein which GDI-bound form in the cell. The role of GDI in transports Rab to geranylgeranyltransferase partitioning G proteins between the mem- II (Fig. 7C) [66]. 840 M. Paduch and others 2001

Figure 7. Guanine nucleotide dissociation inhibitors. A. Rac2×RhoGDI (LyGDI) complex (PDB 1ds6). C-terminal immunoglobulin-like domain is designated LyC, and N-terminal, LyN; linker recognized by ICE , ICE. B. Surface and electrostatic potential of LyGDI. Positively charged (basic) regions in blue and negatively charged (acidic) ones in red. The hydrophobic cavity responsible for binding of the C-terminally attached isoprenyl moiety is also shown. The switch I (SwI) and switch II (SwII) regions are buried in the interaction site. C. RabGDI (PDB 1gnd). The GCD domain responsible for Rab binding is shown.

RhoGDI is composed of 204 amino-acid resi- contains a large and deep (12 ´ 8 ´ 10 Å) dues and contains two domains: a C-terminal isoprene-specific hydrophobic cavity (Fig. 8B) 16 kDa domain that strongly binds the small [68–70]. Docking of the lipid moiety depends G protein through its C-terminal Cys-attached on the presence of an Asp residue in a close lipid moiety and an N-terminal domain, re- proximity to the hydrophobic cavity. Exclud- sponsible for switch region binding and nucle- ing the C-terminus, only a small part of the otide exchange inhibition (Fig. 7A) [67, 68]. Rho molecule contacts GDI [71]. In RhoA The C-terminal domain possesses an immuno- switch II and a fragment of the A3 helix form globulin-like type S fold which seldom occurs the contact interface. The N-terminal domain in cytosolic proteins. The structure is built of in free RhoGDI is basically unstructured and nine b-strands (antiparallel b-sandwich with a sensitive to . It has been shown Greek key topology) and a single 310-helix. that this domain can be proteolytically The domain is highly protease resistant and cleaved by cysteine proteinases that belong to Vol. 48 Small G proteins and their regulators 841

Figure 8. GTPase activating proteins. a × × –× GAPs (coloured helices on the right) complexed with G proteins (C -worm in gray). A. Rho GDP AlF4 RhoGAP × × × × –× (PDB 1tx4). B. H-Ras GDP p120GAP (PDB 1wq1). C. Gia1 GDP AlF4 RGS4 (PDB 1agr). the ICE/CED-3 family (Fig. 7B). Hydrolysis of the accessibility of the geranylgeranyl group the N-terminal LyGDI (a homolog of RhoGDI) to RhoGDI, followed by the step of the gera- domain by IL-1b-convertase (ICE) leads to the nylgeranyl moiety extraction from the mem- inactivation of LyGDI and, therefore, is in- brane by the C-terminal domain of GDI [67, volved in the regulation of inflammatory pro- 73]. cesses and [72]. The N-terminal, dis- ordered, fragment becomes structured upon Rho binding and provides more than 60% of GAP the total Rac×RhoGDI interface making thus a major contribution to the complex binding GAP proteins accelerate the rate of GTP hy- energy. Upon RhoGDI binding to Rho a helical drolysis by four to eight orders of magnitude. hairpin structure is formed that obscures Only a small part of the GAP family has proba- switch I and II [68]. The masking of the bly been recognized and the known members effector region directly prohibits effector include p50, p120, p130, p85, myr5, Bcr, Graf, binding. In the complex, the conformation of , and Abr. Mutational analysis of Ras switch II precludes GTP hydrolysis independ- has shown that the switch regions I and II are ently of GAP presence, and the conformation responsible for the interaction of G proteins of switch I abolishes nucleotide exchange. Ad- with GAP. The interaction forces a confor- ditionally, the Thr residue that coordinates mational change that rigidifies the G domain 2+ Mg efficiently blocks GEF catalyzed nucleo- and promotes a transition towards the g-phos- tide exchange. In the Rho family the surface phate pentacovalent intermediate state of the epitope that binds the N-terminus of RhoGDI hydrolytic reaction [26]. The acceleration of is highly conserved [70]. Two-step kinetics is GTP hydrolysis is possible thanks to the cata- observed for Rho/RhoGDI binding. In the lytic Arg residue supplied by the GAP mole- first step a reorientation of the Rho molecule cule (Fig. 9) [74, 75]. GAPs are predominantly by the N-terminal domain of GDI increases a-helical proteins and sometimes contain a 842 M. Paduch and others 2001

Figure 9. . A. Structure of H-Ras×GDP×p120GAP (PDB 1wq1). p120GAP participates in GTP hydrolysis by insertion of cata- lytic R789 (red) present in the finger loop. Catalytic Q61 (green) (corresponding to Q204 in Gia1) is well oriented in × × – the interaction of switches I and II with p120GAP. B. Structure of Gia1 GDP AlF4 (PDB 1gfi). R178 (red) from switch I directly participates in GTP hydrolysis. four-helix bundle motif (Fig. 8) [76, 77]. Addi- finger helps align a water molecule for tionally they contain flexible regions that can nuclephilic attack together with the catalytic undergo conformation changes upon binding Gln [43]. Association of GAP with the G do- to GTPases [74, 76]. The C-terminal domain of main leads to the rigidifying of the switch re- Bcr (BH) has a number of conservative resi- gions and to entropic stabilization of the inter- dues along a shallow groove formed by two he- mediate state of the reaction [80]. Catalytic lices that are responsible for the interaction residues are oriented and stabilized in a way with the GTPase [78]. that accelarates GTP hydrolysis. Binding of Proteins with GAP activity often contain GAP also stabilizes the residues responsible other domains that are characteristic for cel- for Mg2+ coordination and thus additionally lular signaling processes, such as SH2, SH3, rigidifies the interaction of small G protein PH, and proline-rich regions [79]. The a sub- and GTP [74]. units of heterotrimeric G proteins also pos- sess their own regulatory proteins, called RGS, with a GAP activity. RGS proteins accel- GEF erate about 100 times the rate of GTP hydroly- sis. Unlike GAPs, RGSs only stabilize the In addition to inefficient catalysis, small G GTPase domain since the catalytic Arg resi- proteins are not capable of efficient GDP dis- due is present in the catalytic domain [42]. sociation. GEF proteins accelerate GDP/GTP RGS-like domains are often found in other exchange in small G proteins as a response to types of GTPase regulators. the extracellular signal. Among the already Based on the structural data of small G pro- known GEFs are: Dbl, Vav1, Dbs, Lbc, Lfc, teins and their activating regulator com- Lsc, Vav2, p115RhoGEF, PDZ-RhoGEF, plexes, a mechanism of GAPs action has been Tiam1, FGD1, frabin, Rcc1, cytohesin1, Sec7, proposed. A catalytic Arg residue is located in Gea2p, SOS, Trio, Pix, Mss4, p140RasGRF, a loop region of GAP and forms an argi- and C3G [3]. Chosen GEF structures are nine-finger motif (Fig. 9) [26]. The arginine shown in Fig. 10. Typically, GEFs are large, Vol. 48 Small G proteins and their regulators 843

Figure 10. Guanine nucleotide exchange factor. A.Rcc1×Ran complex (PDB 1i2m). Rcc1 shown in colour as a seven-bladed b propeler [81]. B. The Cdc25 domain of SOS-1 complexed with H-Ras (in gray) (PDB 1bkd) [82]. C. The Sec7 domain of ARNO an exchange factor for Arf (PDB 1pbv) [83]. multidomain proteins that consist of a cata- The RGSL domain (p115RhoGEF) stimulates lytic domain composed of 200–300 amino- GTPase activity in Ga12 and Ga13 and in this acid residues and flanked by other domains way p115RhoGEF acts as a modulator of Ga responsible for oligomerization, protein–pro- regulation of Rho [85–87]. tein and protein–lipid interactions. GEF asso- GEF proteins of small G proteins accelerate ciated domains include: a tandem of DH-PH dissociation of bound GDP and promote the domains (Dbl homology — pleckstrin homo- formation of the GTP-bound state. Some logy), SH2, SH3 (Src homology 2, 3), RGSL GEFs have catalytic activity only towards a (regulators of G protein signaling-like), also single GTPase (for example, FGDI to Cdc42, known as LH (Lsc homology), PDZ, SAM UNC-73B to Rac), while others act on a panel (sterile alpha motif), IQ, C2, CH (calponin of targets (Dbl and Ost both activate Cdc42 homology), proline-rich, and rubredoxin do- and Rho) [88–91]. In nearly all cases the cata- main (two CXXC Zn2+ coordinating se- lytic DH domain is arranged in tandem with quences) [3, 84]. SH2 domains are responsible the about 100 amino acid large PH domain for phosphotyrosine recognition, whereas (Fig. 11). The latter domain is a widely distrib- SH3 recognize proline-rich regions. Both uted signaling module involved in intra- serve as protein–protein interaction adapt- cellular membrane targeting [92, 93]. It has ers. The IQ motif, present in p140RasGRF, is been suggested that in signaling pathways an regulated by complexed with Ca2+. interaction through a PH domain could acti- 844 M. Paduch and others 2001

are two ways of GEF-catalyzed dissociation of GDP: stabilization of the nucleotide-free form of G protein and disruption of the GDP bind- ing site [99]. In the complex, switch I, II and P-loop regions of Ras are in the contact area with SOS. The main interaction site is formed by the switch II region that undergoes a conformational change upon complex forma- tion. The switch I region is weakly ordered in Ras×GDP and is replaced by a helical hairpin of SOS in the complex. Glu942 and Leu939 of SOS protrude to the g-phosphate and Mg2+ binding site. The Glu residue interacts with Ser within the P-loop and destroys contacts Figure 11. Structure of the DH and PH domains. between phosphate binding loop and the Structure of two domains from the human SOS-1 pro- b-phosphate group of GDP. Leu939 abolishes tein (PDB 1dbh). The linker segment and the GTPase Mg2+ coordination sphere and thus decreases binding site are indicated. the affinity to GDP. Such structural distortion moves the P-loop to the position that makes vate GEFs by recruiting them to membranes, nucleotide binding impossible. Generally, the where prenylated small G proteins are located interaction area between GEF and small G [94, 95]. However, it is also possible that the protein is large in order to stabilize the tran- PH domain fulfils a more specific role in regu- sient but otherwise unstable nucleotide-free lating GEF activity by providing catalytic resi- form of the G protein. The G domain without dues, contributing to the GEF/G protein bind- the bound nucleotide is solvent exposed and ing, allosterically modulating the catalytic ac- capable of accepting a GTP molecule and GEF tivity or recruiting additional regulatory fac- dissociation. This step limits the rate of nucle- otide exchange [99, 100]. tors (for example, PtdIns(3,4)P2) [96]. The DH domain of SOS is of predominantly helical structure and comprises 11 helical seg- We thank Dr. Zygmunt Derewenda for help- ments that form a bundle. Three of these heli- ful discussions. cal segments are interrupted by kinks and three other conserved long helices pack to- gether to form a core of the DH domain REFERENCES (Fig. 11) [97]. A typical PH domain fold con- sists of seven b-strands forming a b-sandwich 1. Bourne, H.R., Sanders, D.A. & McCormick, F. flanked at one end by a C-terminal a-helix. (1990) The GTPase superfamily: A conserved Most PH domains contain a phosphatidylino- switch for diverse cell functions. Nature 348, sitol lipids binding site [98]. The DH and PH 125–132. domains are joined by a 13-residue inter - 2. Bourne, H.R., Sanders, D.A. & McCormick, F. domain linker. The interaction between DH (1991) The GTPase superfamily: A conserved and PH occurs mainly via helices, forming a structure and molecular mechanism. Nature tight interface. Interestingly, the regions of 349, 117–127. both domains that form the interface are weakly conserved (Fig. 11) [97]. 3. Takai, Y., Sasaki, T. & Matozaki, T. (2001) Based on the crystal structure of the Small GTP-binding proteins. Physiol Rev. 81, SOS×GDP×Ras complex (Fig. 10B) [97], there 153–208. Vol. 48 Small G proteins and their regulators 845

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